It has become increasingly clear that NK cells can promote DC activation and polarization of the subsequent immune response. This project will evaluate, in transplantable and spontaneous murine tumor models, the significance of NK-DC interactions for the generation and function of anti-tumor CTLs and will improve antitumor activity against established MHC+/MHC- tumors by controlling/manipulating these interactions. We hypothesize that activated NK (A-NK) cells, delivered to tumor sites, will kill tumor cells and thereby liberate both mediators of acute inflammation (such as HSP and HMGB1) and antigens which can be taken up by DCs. In addition, contact- and cytokine-dependent cross-talk between DCs and NK cells will further activate both cell types, leading to the generation of polarized DCs with enhanced capability of stimulating Th1-anti tumor responses. This will be evaluated in three animal models: 1) a model tumor antigen expressing system (the MO-5, ovalbumin transfected B16 melanoma). Effective NK /DC interactions will, in this model be evaluated by measuring the number of adoptively transferred ova-specific OT-I (CDS) and OT-II (CD4) cells in tumors, lymph nodes, blood and spleen; 2) The wild-type B16 model and; 3) in the p16INK4a-/-KO model of spontaneous melanoma. Following systemic or local delivery of A-NK cells and DCs, we will vary the cytokine profile in the tumor microenvironment. To achieve this, DCs and NK cells will be transduced with cytokine gene-containing viral vectors to produce various pro-NK and pro-DC cytokines, e.g., IL2, IL18, IFN-alpha and GM-CSF. In addition, the ability of multiple NK/DC injections to ensure continuous stimulation of the afferent limb (and thereby resultant CTL generation) as well as local CTL effector function, will be tested. Three Specific Aims will be conducted to explore these issues: Aim 1. Define the in vivo survival of NK cells and DCs as well as their traffic to tumors and lymph nodes following injection different NK/DC ratios into tumor-bearing animals via different routes. Aim 2. Demonstrate that intratumoral delivery of NK cells and DCs promotes CTL generation and function in transplantable and spontaneous tumor models.; Aim 3. Enhance NK/DC function in tumors by forced cytokine-gene transduction of NK cells and DCs.